Genetic insights into Cyphocharax magdalenae (Characiformes: Curimatidae): Microsatellite loci development and population analysis in the Cauca River, Colombia

Cyphocharax magdalenae, a Colombian freshwater fish species, plays a vital role in nutrients distribution and serves as a significant food source for other fish species and local fishing communities. Considered a short-distance migratory species, C. magdalenae populations face substantial extinction risk due to human activities impacting their habitats. To address the lack of knowledge on genetic diversity and population structure, this study used next-generation sequencing technology to develop species-specific microsatellite loci and conducted a population genetics analysis of C. magdalenae in the middle and lower sections of the Cauca River, Colombia. Out of 30 pairs of microsatellite primers evaluated in 324 individuals, 14 loci were found to be polymorphic, at linkage equilibrium and, in at least one population, their genotypic frequencies were in Hardy-Weinberg equilibrium. Results showed high genetic diversity levels compared to other neotropical Characiformes, with inbreeding coefficients similar to those reported for phylogenetically related species. Moreover, C. magdalenae exhibits seasonal population structure (rainy-dry) consisting of two genetic stocks showing bottleneck signals and high effective population sizes. This information is essential for understanding the current species genetics and developing future management programs for this fishery resource.

This study was supported by a grant framed under the Project "Variabilidad genética de un banco de peces de los sectores medio y bajo del río Cauca" (CT-2019-000661, Empresas Públicas de Medellín and Universidad Nacional de Colombia, Sede Medellín).Funders do not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript Please select the country of your main research funder (please select carefully as in some cases this is used in fee calculation).

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Introduction
Curimatidae family encompasses 8 genera and 119 valid species distributed in South America (Fricke et al., 2023).Cyphocharax Fowler 1906 genus is one of the most abundant having 47 valid species (Fricke et al., 2023) and wide distribution ranging from the Pacific rivers in southern Costa Rica to the La Plata river and various coastal drainages in central Argentina (Maldonado-Ocampo et al., 2008;Melo et al., 2018).It has been found that Atlantic basins have a much more diverse group expanding from the Orinoco river, the Amazonas river, rivers from the Guayanas and eastern Brazil; on the contrary, a less diverse group of species habits the rivers in northern Peru to Costa Rica, comprising the tributaries of Magdalena and Maracaibo (Vari et al., 2010).
Cyphocharax magdalenae, listed as a Least Concern species by The International Union for Conservation of Nature (IUCN), has been gaining fishing importance during the last years due to the replacement of captured traditional species as catfish Prochilodus magdalenae Steindachner 1879 and Blanquillo Sorubim cuspicaudus Littmann, Burr & Nass 2000 (Blanco et al., 2005;Jimenéz-Segura & Lasso, 2020); nevertheless, its notable mortality because of overfishing suggests a possible decrease in its populations (Lasso et al., 2011;Duarte et al., 2019Duarte et al., , 2021)).
The latter is relevant since currently C. magdalenae lacks genetic studies that allow knowing the present status of the species and delving into its ecological processes, which hinders the development and application of plans for the protection and conservation of the species.This scarcity in information contrasts with population genetic studies performed for Curimata mivartii Steindachner  (Gallo & Díaz-sarmiento, 2003;García-Castro et al., 2021), Brycon henni Eigenmann 1913 (Pineda et al., 2007;Hurtado-Alarcón et al., 2011;Landínez-García & Márquez, 2020) Carvajal-Escobar, 2020;Cerón et al., 2021), has great economic importance because of its influence in most prioritized productive chains in Colombia, which include small exploitations, extensive industries, monocultures, energy generation, mineral extraction, agriculture and fishing (Carvajal et al., 2016;González-López & Carvajal-Escobar, 2020;Jimenéz-Segura & Lasso, 2020).This has caused that fishes are one of the most threatened biological resources, with little-or unknown local priorities over the rivers (Corporación Autónoma Regional de Cundinamarca (CAR), 2012; Hernández- Barrero et al., 2021).

Biological material and study area
This study analyzed a total of 324 muscle tissues and/or fins of C. magdalenae preserved in ethanol 70%, provided between 2019 and 2021 by Universidad de Antioquia, Universidad de Córdoba, and Universidad Nacional de Colombia Sede Medellín, through scientific cooperation agreement CT-2019-000661, framed within the environmental license # 0155 of 30 January 2009 of Ministerio de Ambiente, Vivienda y Desarrollo Territorial for the construction of hydroelectric Ituango.Samples from individuals were collected in 19 sites of the main channel, some swamps, and tributaries of the lower sector of the Cauca river.This zone, characterized by a large alluvial plain surrounded by mountains and flat and wavy surfaces wherein various swampy complexes are formed (Mejía et al., 2007;Betancur et al., 2009), has been exposed to different anthropic activities including stockbreeding, fishing and extensive cultures (Corantioquia -Universidad de Antioquia, 2014).
Additionally, high levels of mercury and sediments are reported, as well as elevated amounts of vegetations in the main courses of the river as a result of deforestation and flooding that may affect

Microsatellite loci identification and development of primers
Methodology described by Landínez-García & Márquez (2016Márquez ( , 2018) ) was followed for developing species-specific microsatellite loci for C. magdalenae.The genomic library was built from total DNA extraction from the spin (5,775ug) of a C. magdalenae individual captured in the Cauca river (CYPHO11886) using the DNA extraction reagents and recommendations of the manufacturer PureLink genomic DNA Mini Kit (Invitrogen), then it was used for NGS surface sequencing (Whole Genome Sequencing), through Illumina Miseq (300 PE).After cleaning the raw reads using PRINSEQ lite (removal of adapter sequences utilizing <Q30 quality bases), 100,000 high-quality extended reads were analyzed with PAL_FINDER v0.02.03 (Castoe et al., 2010) for extracting reads containing tri-, tetra-and pentanucleotide microsatellite motifs.Then, Primer3 v2.0 (Rozen & Skaletsky, 2000) was used for the primer designs in the flanking sequences of the microsatellite loci and ultimately the correct evaluation of the primers was tested with ePCR (Rotmistrovsky et al., 2004).Selection of the set of the microsatellite loci was performed according to already stablished features for validating new primers of microsatellite loci (Fernandez-Silva et al., 2013;Schoebel et al., 2013).
For genotyping, 10μl reaction mixtures were used with final concentrations of 0.3pmol/μl of each forward primer tagged on the 5' end with one of the adapters (tails A, B, C and D) described by Blacket et al., 2012, 6pmol/μl of each reverse primer, 0.5pmol/μl fluorescently labeled adapter (6-FAM, VIC, NED and PET, Applied Biosystems), 1X Master Mix, 2.5% v/v GC Enhancer Platinum Multiplex PCR Master Mix (Applied Biosystems) and 3-5μg/μl DNA isolated using the Purelink® purification kit (Thermo Scientific) with a modification in digestion time of 24 h when working with fins.Thermal profiles included an initial denaturalization step of 90 °C for 35 s and an annealing step of 56 °C for 35 s (with no final elongation).Subsequently, amplicons were separated by electrophoresis on an ABI 3730 XL automated sequencer using 600LIZ as the internal molecular size.
For detecting atypical loci and determining evolutive forces acting over the microsatellite loci, BayeScan v2.1 (Foll & Gaggiotti, 2008) was used for performing an analysis employing the parameters for prior odds of 10:1 for the neutral model, 20 pilot runs, each having 5,000 iterations, followed by 100,000 iterations and a burn-in of 50,000.Critical values were based on the posterior probability of the Bayes factor, following the Jeffrey's scale (Nagin, 1999), which sets a probability of 0.76 as substantial evidence for selection.
To determine recent genetic bottlenecks, excess heterozygosity was tested under the mutation drift equilibrium assumption in three mutational models of the microsatellite loci (IAM: infinite alleles model, SMM: stepwise mutational model, TPM: two-phase model) through Wilcoxon signed rank test (Luikart & Cornuet, 1998) included in Bottleneck v.1.2.02 (Piry et al., 1999).Moreover, M ratio (mean ratio of the number of alleles to allele size range; Garza & Williamson, 2001) included in Arlequin v3.5.2.2 (Excoffier et al., 2005) was calculated following the criteria according to which values lower than 0.68 indicate recent and severe reductions in population size (Garza & Williamson, 2001).Furthermore, effective population size (Ne) of the species was determined in each of the assessed populations using the linkage disequilibrium method and a minimum allele frequency of 0.02 implemented in NeEstimator v2.1 (Do et al., 2014).Change in HE of the species in 10, 50 and 100 generations (t) was estimated based on the equation proposed by Crow & Kimura, 1970 (/0 = [1-1/(2)]  ).Genetic deterioration grade was stablished using the critical values of 25% Ht reduction in 10 (critically in danger), 50 (in danger) and 100 (vulnerable) generations (Willoughby et al., 2015).

Genetic structure
Bayesian analysis of population partitioning in STRUCTURE v.2.3.4 (Pritchard et al., 2000) was performed to determine samples grouping according to their co-ancestry coefficient.Parameters included 1,000,000 Monte Carlo Markov steps; 100,000 iterations were dismissed as burn-in to estimate each K value (1 -8).Each analysis was repeated 20 times.For a best estimation of genetic stocks (K), STRUCTURESELECTOR (Li & Liu, 2018), was used to calculate the ΔK ad hoc statistic (Evanno et al., 2005), estimators MEDMEANK, MAXMEANK, MEDMEDK and MAXMEDK (Puechmaille, 2016), and to graphically represent the results using the integrated CLUMPAK software (Kopelman et al., 2015).
Additionally, a discriminant analysis of principal components (DAPC) was performed in polymorphic loci genotype resulting in 324 analyzed individuals using the R-package Adegenet (Jombart, 2008).
A group of 16 out of 30 tested loci showed clearly defined peaks and absence of stutter bands in the electropherograms; these microsatellite loci include tri-(3) and tetra-(13) nucleotides motifs.Micro-Checker analysis did not indicate stutter associated genotyping errors nor an allele loss in the loci (dropout) and linkage disequilibrium tests were not significant.Departures of allele frequencies from Hardy-Weinberg equilibrium were identified as well as heterozygote deficits (Table 1).In general, microsatellite loci exhibited allele size between 106 and 414 bp with PIC values ranging from 0.733 to 0.954 and HO and HE between 0.408-0.916and 0.762-0.957,respectively (Table 1); no evidence of atypical loci was detected by Bayescan.Nonetheless, null alleles evidence was found in loci Cym10 and Cym16, for which they were excluded in subsequent analysis.

Genetic diversity and demographic events 233
Genetic diversity estimators (Table 2) showed that S7 had the lower average number of alleles per 234 locus (15.714 alleles/locus) while S5 exhibited the higher mean number (18.857 alleles/locus).Mean 235 HO was lower in S7 (0.772) and higher in S8 (0.827) and mean HE displayed the lower value in S5 236 (0.880) and the higher in S4 (0.887).Only two loci (Cym8 and Cym14) out of the 14 selected departed 237 from the Hardy-Weinberg equilibrium in four of the five evaluated sites and the remaining fulfilled 238 the assumption in at least two sites.A similar result of genetic diversity was observed in the genetic 239 stocks (Stock1 and Stock2, Table 2) found by the genetic structure Bayesian analysis, as shown 240 below.
Moreover, multiloci values (across loci) of the inbreeding coefficient for each of the populations showed statistical significance with greater impact in population S7 (FIS=0.120,P=0.000).These values, although remained significant, significantly decreased in the stocks (FISStock1=0.051,P=0.000; FISStock2=0.071,P=0.000).It was found that the assessed populations recently suffered a drastic reduction in population size since both the modified Garza-Williamson index (M-ratio: 0.238-0.256)and P values of the Wilcoxon signed rank test for the IAM and the TPM (except from one site) showed statistical significance (Table 3).Results for SMM, however, were not significant.Furthermore, the effective size calculation in each of the sampled sites showed the lower values in S4 and S7 and the higher in S6 and S8; nevertheless, it was not possible to obtain a value for S5, and estimation was obtained only for one of the genetic stocks (Stock1) (Table 3).Due to the latter, in subsequent analysis the lower limits of confidence intervals were used for S5 and Stock2.
For all evaluated generations, the species is classified as non-threatened as no reduction percentage reached 25% (critical value of the classification) (Table 4).S8 showed the lower reduction percentage in all generations (0.002, 0.011, 0.021); nevertheless, S7 and S4 exhibited the greater loss of heterozygosity percentage in 100 generations with 20.034 and 14.645, respectively.Moreover, in the evaluation on genetic stocks no approximation surpassed the 5% reduction in all generations; in fact, similar values were found in each stock for each generation, although Stock1 showed slightly higher values.

Genetic structure
In approximations set for determining population structure, it was found that the most backed group by all methods used in the Bayesian inference is a ΔK = 2.These two groups coexist in all sampled sites as confirmed by the STRUCTURE graphic (Fig 2A).Additionally, it was observed that in sectors S4 and S5 predominates Stock1 (pink) while Stock2 (purple) is predominant in sectors S6, S7 and S8.This is corroborated by the discriminant analysis since it showed that the two genetic stocks were not homogeneously distributed (Figs.2B, 2C); only one similarity was found in the distribution of the tested individuals in sites S6 and S7 (Fig 2B).Differences in the frequencies of the stocks agree with results obtained in AMOVA (F´ST= 0.003, P = 0.001) and the pairwise comparisons of the standardized statistics F'ST, DEST (Table 5), which showed significant differences between S8 in comparison to the remaining sites, and S6 in relation to S4 and S5.

Discussion
This study tested three hypotheses related to population genetics of C. magdalenae: (i) low levels of genetic variability, high levels of consanguinity and recent bottlenecks, probably as a result of alterations in its habitat due to different anthropogenic activities, (ii) gene flow because of scarce or non-existent geographic barriers of its habitat and (iii) seasonal population structure following the bimodal behavior of the Cauca river.To contrast these hypotheses, this study developed a set of 30 microsatellite loci of neotropical fish C. magdalenae of Colombia; 16 out of which having tri-and tetranucleotide repetition motifs showed to be polymorphic, highly informative and with ability to detect diversity levels and genetic structure in the low section of the Cauca river.
The selected microsatellite loci were preferably those having tri-and tetranucleotide repetition motifs as these repetition patterns are the most recommended due to their simplicity for genotyping and allele classification (Castoe et al., 2010(Castoe et al., , 2012;;Guichoux et al., 2011).All microsatellite loci exhibited PIC values that allow describing them as highly informative according to Bostein et al., 1980;moreover, these (Macias et al., 2009).
Furthermore, the hypothesis of high levels of consanguinity in C. magdalenae was not confirmed since the species showed inbreeding signals below 10% in each genetic stock (FIS Stock1=0.051;FIS Stock2=0.071).Although these values do not exceed the proposed limit (Franklin, 1980;Soule, 1980), it has been indicated that any inbreeding coefficient greater than cero has unfavorable effects on the biological efficacy (Frankham et al., 2014) (Ribolli et al., 2017).
Furthermore, this study supports the hypothesis of a recent genetic bottleneck in C. magdalenae.
These results may be a consequence of the joint action of various anthropogenic activities impacting the environment or directly affecting the C. magdalenae individuals, or climatic changes.For instance, for The IUCN, C. magdalenae shows a decrease in its populations due to the fishing pressure (Lasso et al., 2011;Duarte et al., 2019Duarte et al., , 2021)), which is consistent with the reduction reports for the species in the Magdalena basin with a diminishment in 887.7 tones unloaded for 2019 (Duarte et al., 2019) in comparison to 436,1 tons for 2021 (Duarte et al., 2021).Moreover, the species does not have a recommended (R) minimum capture size nor suggested by any in force regulation (N) (AUNAP-UNIMAGDALENA, 2013); in fact, it was found that between 2018 -2021 ranges of capture size were lower than those described for size at maturity (Duarte et al., 2021).As it has been proposed for other species (Restrepo-Escobar et al., 2021), contamination with products derived from mining, could be affecting the viability of C. magdalenae, which is supported by evidence of methylmercury in fishes collected in the zone (Cruz-Esquivel & Marrugo-Negrete, 2022).Another threaten is the establishment of introduced exotic species (Lasso et al., 2011), such as Basa fish Pangasianodon hypophthalmus (Sauvage 1878), omnivorous species of fast growth in size and weight, and extension in Colombia mainly in the Magdalena-Cauca basin (Corporación Autónoma Regional de Cundinamarca, 2019), and the Nile Tilapia Oreochromis niloticus (Linnaeus 1758), which has survival ability in all types of habitats, from salty, marine waters to estuarine and continental waters with temperatures between 8 and 42 °C (Corporación Autónoma Regional de Cundinamarca, 2018).
Despite the introduction of O. niloticus in Colombia was done with commercial purposes and control of Oreochromis mossambicus (Peters 1852), an aggressive behavior against native species P.
magdalenae has been displayed (Rico, 2010) and, after its introduction, there has been a reduction in the abundance of Triportheus magdalenae (Steindachner 1878) in the Guajaro reservoir in the Magdalena (Caraballo & Gandara, 2009).
In addition to the anthropogenic activities, it has been documented that the impact of climate factors has provoked changes in fish populations as they are responsible for abrupt changes in environments such as high temperatures, alterations of hydrological cycles, reduction of dissolved oxygen, changes in mortality rates, growth, reproduction and distribution of fish populations (Serna-López & Cañón-Barriga, 2020).This climate factor has also been used for explaining population genetics aspects of C. mivartii (Landínez-García et al., 2016) and P. magdalenae (Landínez-García et al., 2020).
Contrary to the bottleneck evidence, estimations performed over the effective population size (Stock1: 1464.6,Stock2: 2127) were higher than the critical value (Ne ≤ 1000), which allows deducting that the species has long term maintenance (Frankham et al., 2014).This idea is also supported by the results of conservation status as even in 100 generations (260 years: species with generational time of 2.6 years; Puentes et al., 2014) estimations did not show evidence of reductions in heterozygosity greater than 25% (critical value of the classification), for which the species can be listed as non-threatened.This classification, however, should be interpreted carefully since it has been indicated that between 4-10% of freshwater species in South America are exposed to a certain risk of extinction mainly due to habitat loss or degradation (Reis et al., 2016).
To conclude, this study allowed determining that C. magdalenae in the low section of the Cauca river exhibits seasonal population structure formed by two genetic stocks associated to the rainy and dry seasons, and which show high genetic diversity, low inbreeding indexes, bottleneck signals and large effective population size.Data obtained along with the microsatellite loci developed de novo in this study are a starting point for future research directed to the monitoring of the genetic diversity and population structure of this species to develop proper management plans.
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Fig 2 .
Fig 2. Results of Bayesian inference of structure (a) and discriminant analysis of principal components of Cyphocharax magdalenae: (b) Five sites evaluated (c) Two genetic stocks.

Table 3 . Bottleneck tests and effective population size (Ne) of Cyphocharax magdalenae in the five sampling sites and genetic stocks of the Magdalena-Cauca basin. Statistical significance is noted in bold (P<0.05).
1 P values of the Wilcoxon signed rank test of one tail in IAM, SMM and TPM; 2 M-ratio < 0.68 denote recent reductions in the population; 3 IC 95% using the JackKnife method(Waples & Do, 2008).* Statistical significance.
Considering that this is the first study performed for Cyphocharax genus, high diversity was found in comparison to species of the same family as C. mivartii (Na: 10.493, HO: 0.757, HE: 0.801; Landínez-García & Marquez, 2018) with whom it shares its habitat.Likewise, when comparing these results with species of phylogenetically related families that also inhabit the Cauca river, C.
(Márquez et al., 2020)uez, 2018)those reported for Characiformes in the Magdalena-Cauca basin using species specific microsatellites, C. magdalenae exhibited levels of inbreeding relatively similar to those reported for C. mivartii (0.040 -0.087;Landínez-García & Marquez, 2018); lower values to those described for P. magdalenae(0.125 -   0.255; Landínez-García et al., 2020)and greater to those found in Brycon henni (-0.040 --0.009;Landínez-García & Márquez, 2020), a phylogenetically farther species.Additionally, this study corroborated the hypothesis of gene flow despite having found significant differences among the individuals of the lower sector of the Cauca river as said differences are related to the coexistence of two genetic stocks with uneven distribution through the river.This indicates that there is no evidence of barriers separating the gene flow of C. magdalenae in the low section of the Cauca river, which is consistent with data reported for C. mivartii (Landínez-García & Marquez, 2018), P. magdalenae(Landínez-García et al., 2020), Cynopotamus magdalenae (Steindachner 1879), Megaleporinus muyscorum (Steindachner 1900)(Márquez et al., 2020)and catfishes A.
(García- Castro et al., 2021)1)spicaudus(Restrepo-Escobar et al., 2021), P. magadaleniatum(García- Castro et al., 2021), P. atricaudus and P. magnus(Rangel-Medrano & Márquez, 2021).Nonetheless, genetic structure due to seasonal variation was found since comparisons over the samples in rainy and drought periods during the assessed years allowed detecting that a stock predominates in the dry season (stock 1) while the other stock predominates in the rainy season (stock 2) (Figs.3A, 3B).Sampling in both seasonsfor two years reflects an alternated cyclic behavior between both stocks and greater proliferative advantage of Stock2 over Stock1, which suggests a differential reproductive success leading to different cohorts (temporal Wahlund effect).These results are similar to those reported by Da Rosa et al., 2022 for Prochilodus lineatus (Valenciennes 1837) in the Mogi-Guaçu river in Brazil as the individuals analyzed between 2005 and 2006, in rainy and dry seasons (January and August, respectively) showed evidence of temporal genetic structure.Furthermore, it was found that Salminus brasiliensis (Cuvier 1816) in the Uruguay river in Brazil is formed by three genetic stocks resulting from a temporal genetic structure in which seasonal precipitations in the river are one of the main factors that could have originated genetic groups as the water level is fundamental in reproductive migration